环介导等温扩增技术检测犬利什曼原虫感染方法的建立

发布时间：2018-03-28 17:08

本文选题：婴儿利什曼原虫　切入点：动基体小环DNA　出处：《中国疾病预防控制中心》2013年硕士论文

【摘要】：目的利什曼病是由利什曼原虫无鞭毛体寄生在包括人类在内的哺乳动物巨噬细胞而引起的人兽共患寄生虫病。在我国当前流行的6省(区)中,有四省为动物源型,犬是主要传染源。对犬的管理是控制此类型黑热病的关键。因此,研究有效的快速简便适于现场使用的检测犬利什曼原虫感染的方法对控制该型黑热病具有重要意义。利什曼原虫动基体DNA (kinetoplast DNA, kDNA)是由10-20个拷贝的大环和上万拷贝的小环构成,小环序列不仅在种间,而且在种内,甚至在株内均存在高度异质性。因此,小环成为特异敏感检测利什曼原虫感染极好的靶分子。环介导等温扩增技术(1oop-mediated isothermal amplification, LAMP)具有灵敏度高、操作简单快速以及对仪器设备依赖性低的特点。本研究的目的是建立起以犬眼结膜为检测样本,以kDNA小环为检测靶分子的环介导等温扩增技术,从而为检测我国犬利什曼原虫感染提供敏感、特异、快速、简便、适合于现场应用的检测方法。方法应用来自利什曼原虫kDNA小环保守区的一对特异性引物KP1/KP2扩增我国利什曼原虫四川株小环DNA全长序列并测序,对序列进行分析；应用获得的序列在线设计多组LAMP引物,依据文献设立初始反应体系和扩增条件对每组引物的反应性进行评价,选取一组能进行敏感、特异扩增的引物应用于LAMP方法建立。应用选取的引物确定影响LAMP扩增的关键因素Mg2+和dNTPs最适浓度以优化反应体系,比较浊度法、SYBR Green法和钙黄绿素法3种观察反应结果的方法的效果,对建立的LAMP法的敏感性和特异性进行实验室评价。应用建立的LAMP方法检测现场样品(来自动物源型内脏利什曼病流行区和非流行区犬眼结膜拭子,煮沸后直接作为扩增模板),并与骨髓镜检和静脉全血常规PCR检测结果进行比较。结果应用KP1/KP2引物从汶川株利什曼原虫扩增出约850bp的动基体小环DNA序列。对随机选取的20个阳性克隆进行测序,并对序列进行了比对分析,显示这些序列可区分为两大类,其中以CQ18为代表的15个序列可归为一大类,占比75%,为优势序列,序列长度为858bp,其余以CQ07为代表的5个序列可归为一类,占比25%,序列长度为854bp,CQ18和CQ07两者序列的一致性为82.7%。进化分析显示这两类序列与杜氏利什曼原虫种团的原虫亲缘关系最近。应用CQ18序列进行在线引物设计,得到5组引物,对5组引物分别进行LAMP反应评价,只有第2组引物能引起特异、敏感LAMP反应；对反应体系进行优化,显示Mg2+最适浓度为8mM, dNTPs最适浓度为1.4mM.应用3种方法观察LAMP反应结果,显示钙黄绿素法能显著区分阳性和阴性LAMP反应结果。建立的LAMP法的检出下限为10-7个原虫/mL,而常规PCR采用RV1/RV2和K13A/K13B两对引物,检出下限分别为10-3和10-2个原虫/mL。应用其他8种不同虫种(株)的利什曼原虫kDNA进行LAMP反应,只有来自我国的3株婴儿利什曼原虫虫株JS5、甘犬和801显示阳性反应。应用建立的LAMP法、镜检法和常规PCR法检测内脏利什曼病流行区犬样本,阳性率分别为59.46%(66/111)、7.62%(8/105)和48.1%(53/110),LAMP和PCR阳性检出率无统计学差异(X2=2.83,P0.05)。应用PCR和LAMP检测内脏利什曼病非流行区犬样本,分别出现1(1/33)和2(2/32)例假阳性,LAMP和PCR阴性检出率无统计学差异(X2=0.0007,P0.05)。结论基于我国婴儿利什曼原虫四川汶川株动基体小环DNA序列设计引物,成功建立了以犬眼结膜组织为检测样本的检测我国犬利什曼原虫感染LAMP检测方法,与常规PCR法比较,检测的特异性相当,而检测的敏感性更高,且取样简便,反应快速,适合在现场应用。两种分子方法检测结果均显示我国动物源型黑热病疫区犬利什曼原虫感染率相当高,应加强犬的管理以控制当地的黑热病。
[Abstract]:objective
Leishmaniasis is caused by Leishmania amastigotes parasitic in mammals including human macrophages caused by parasitic disease. In 6 provinces of China, the current popular (area), there are four provinces for animal source, dogs are the main source of infection. The dog management is the key to control this type of leishmaniasis. Therefore study on fast, simple and effective method which is suitable for field use for detection of canine Leishmania infection has important significance to control the type of leishmaniasis. Leishmania kinetoplast DNA (kinetoplast DNA kDNA) is a copy from 10-20 copies of the big ring and tens of thousands of small, small series not only among the species and within species, and even within a tree are highly heterogeneous. Therefore, become small molecular target specific and sensitive detection of Leishmania infection. Excellent loop mediated isothermal amplification (1oop-mediated isothermal amplification, LAMP) has the advantages of high sensitivity, simple operation and rapid equipment dependent low. The purpose of this study is to establish a canine conjunctival as samples to kDNA ring for detecting target molecules guide loop mediated isothermal amplification technology, so as to provide sensitive, for the detection of our canine Leishmania infection specific, fast. The detection method is simple, suitable for field application.
Method
A pair of specific primers of KP1/KP2 from Leishmania kDNA small environmental protection area of our country keep amplification of full-length DNA sequence of Sichuan strain of Leishmania ring and DNA sequencing, sequence analysis; multiple sets of LAMP primers obtained online application design based on the literature, the establishment of the initial reaction system and evaluate the response of each primer amplification conditions, selection a group of sensitive primer application specific amplification in LAMP method. Primers were selected to determine the key factors affecting the application of LAMP amplification of Mg2+ and dNTPs concentration in order to optimize the reaction system, comparative turbidity method, SYBR method and Green method of calcein method 3 to observe the reaction results of the laboratory evaluation of effect. The established LAMP method. The sensitivity and specificity of the LAMP method to detect field samples (from animal sources of visceral leishmaniasis endemic and non endemic areas in dogs The eye conjunctival swabs were directly used as the amplification template after boiling, and compared with the results of bone marrow microscopy and total venous blood routine PCR.
Result
Application of KP1/KP2 primers amplified kinetoplast minicircle DNA sequence of about 850bp from Wenchuan. Leishmania spp. sequencing of 20 positive clones were randomly selected, and the sequence was compared and showed that these sequences can be divided into two categories, including 15 sequences represented by CQ18 can be classified as a class, accounting for 75%, as the dominant sequence, sequence length of 858bp, the remaining 5 sequences represented by CQ07 can be classified as a class, accounting for 25%, the length of the sequence of 854bp, CQ18 and CQ07 of the two consensus sequences for phylogenetic analysis of 82.7%. recently showed that these two kinds of sequence and Liz Leishmania species groups from genetic protozoa application of CQ18 series online. Primer design, 5 sets of primers, 5 primers of group were evaluated in LAMP, only second sets of primers can cause specific and sensitive LAMP reaction; the reaction system was optimized, the optimum Mg2+ concentration is 8mM, dNTPs The optimum concentration of 1.4mM. was observed in the LAMP reaction results of application of the 3 methods, show the calcein method can significantly distinguish between positive and negative LAMP results. The detection limits of the LAMP method established for 10-7 protozoa /mL, while conventional PCR and K13A/K13B RV1/RV2 using two pairs of primers, detection limits were 10-3 and 10-2 protozoa /mL. application the other 8 different species (strains) of Leishmania kDNA LAMP reaction, only from the 3 strains of baby Lishman parasite strains JS5, Gansu dogs and 801 showed positive reaction. The LAMP method is used to establish the microscopic method and conventional PCR method to detect canine visceral leishmaniasis endemic area of samples, the positive rate was 59.46% (66/111), 7.62% (8/105) and 48.1% (53/110), LAMP and PCR positive rate showed no significant difference (X2=2.83, P0.05). The application of PCR and LAMP detection of visceral leishmaniasis in non epidemic area of canine samples, there were 1 (1/33) and 2 (2/32) false positive, L The negative detection rates of AMP and PCR were not statistically significant (X2=0.0007, P0.05).
conclusion
Based on Sichuan Wenchuan strains of Leishmania infantum kinetoplast minicircle DNA primers were successfully established to detect canine conjunctival tissue samples for detection of canine Leishmania infection in China LAMP detection method, compared with the conventional PCR method, the specificity of detection, and detection sensitivity is higher, and the sampling is simple, quick response, suitable for application in the field. The results of the two methods of molecular detection showed animal China black fever epidemic areas of canine Leishmania infection rate is very high, we should strengthen the management of dogs to control the local black fever.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R382.22;R3416

【参考文献】